Production of pantothenic acid



Patented May 15, 3.945

PRODUCTION OF PANTOTHENIC ACID Sidney H. Babcock, Jr., Detroit, Mich., assignor to The Regents of the University of California, Berkeley, Calif., a corporation of California No Drawing. Original application July 19, 1940,

Serial No. 346,454. Divided and this application May 8, 1941, Serial No. 392,536

7 Claims.

This invention relates to a process for coupling lactones such as alpha-hydroxy-beta, beta-dimethylbutyrolactone with amine acids such as beta-alanine or their derivatives, to produce physiologically active substances having a vitamin-like action, such as panthothenic acid, or its derivatives.

Up to the time of the present disclosure there has been no known way of synthesiing pantothenic acid. This compound has been known to exist in the natural state, but it has not been isolated, and knowledge of its existence in the past was only gained by measuring the biological effect of concentrates of pantothenic acids prepared from natural sources such as liver. It is now possible, by carrying out the present disclosure, to prepare it synthetically, thus making it possible to produce this substance as needed and on a large and improved scale.

In accordance with the present invention, in its preferred form, an aqueous solution containing an anion of a potential salt of beta-alanine is reacted with an alpha-hydroxy-beta, beta-dimethylbutyrolactone in the presence of a water soluble base adapted to furnish a hydroxyl ion, the concentration of the reacting ingredients being such as to insure the production of a maximum amount of the salt of pantothenic acid, while keeping the amount of unreacted ingredients to a minimum.

There is also present, a constituent adapted to furnish the positive member of the salt of pantothenic acid. After the reaction has been completed, there is added an additional quantity of a water soluble base, functioning to cause the unreacted beta-alanine to react with the excess lactone and produce an additional amount of the salt of panothenic acid,

This procedure is repeated in order to insure a yield of at least ninety percent of the salt of pantothenic acid,

In the best form of the invention, there is an excess of the lactone and the concentration of the lactone in the reaction mixture is such as to insure the production of a maximum amount of the salt of pantothenic acid, while keeping the amount of the unreacted beta-alanine to a minimum.

The hydroxyl ion is preferably furnished by an alkali or an alkaline earth metal. The most satisfactory results are obtained by using sodium or potassium hydroxide.

While it is desirable that the water soluble base also furnish the constituent having the positive member of the salt of pantothenic acid, this is not necessary.

In the preferred form of the invention, the reactions are carried out in the cold in order to inhibit or minimize the occurrence of side reactions reducing the yield, and the ratio of the sodium hydroxide.

In accordance with this invention, salts of pantothenic acid and pantothenic acid may be synthesized in the following manner: Betaalanine; l N sodium hydroxide; and alpha-hydroxy-beta, beta-dimethylbutyrolactones; in a 1:113 molecular ratio, are mixed in that order at 0 C., and allowed to stand one hour. An amount of sodium hydroxide equivalent to the amount of free beta-alanine remaining in the,

solution (as determined by Sorenson formol titration) is added. After standing 9, second hour, the process is repeated, and is continued until ninety percent or more of the beta-alanine has been converted to pantothenic acid. The pantothenic acid content is then measured by biological assay with chicks, according to the method published in the Journal of Biological Chemistry, volume 117, page 11 (1937), or by other methods of biological assay, as by the use of micro-organisms. By using more concentrated alkali, hence less water, it has been possible to get a sixty per cent total conversion in the first cycle, and a ninety-five per cent conversion in the second cycle,

Alpha-hydroxy-beta, beta-dimethylbutyrolactone has what is termed an asymmetric carbon atom. Because of this, it exists in two enantiomorphic forms and also in a fifty-fifty mixture of the two forms. These three forms are referred to as:

d-alpha-hydroxy-beta, beta dimethylbutyrolactone l-alpha-hydroxy beta, beta-dimethylbutyrolactone dl-alpha-hydroxy-beta, beta-dimethylbutyrolactone In this work there has been used the d1 form (i. e., the mixture) but the same things would all be equally true of the dand lforms separately. This is important, as only one of the two enantiomorphic forms results in physiologically active pantothenic acid. Hence when the dl lactone is used, only half the product is vitaminthe dpantothenic acid; l-pantothenic acid is not vitamin and has no activity. Dl-pantothenic acid is half as active as d-pantothenic acid. But since Just as well with the present method "will work three are specifithe d-, land dl-lactones, all cally mention Many chemical reactions are of the equilibrium type, represented by the following equations:

When any two reactants A and B iorm two products C and D, it frequently happens that the products can inter-react to re-iorm the reactants. A condition of equilibrium results wherein, for every pair or reactant molecules forming product molecules, a pair of product molecules form reactant molecules. The point at which this equilibrium lies is dependent upon the nature oi the molecules involved. This point might be anywhere between the two points where (a) The amount of the reactants left when equilibrium is established is negligibly small, and

(b) the amount of products formed is negligibly small.

0! the various methods of shifting an rium, two will be mentioned.

(a) An excess of one of the reactants can be used. By this means, a greater percentage of the other reactant is converted into product molecule. This is practically useful whenever one of the reactants is cheap and the other expenslve.

(17) One oi'the products can be removed as fast as it is termed. This is a corollary to (a), for it diminishes the amount oi one of the products, and continually upsets the equilibrium, until finally all or most 01' the reactants are converted to products. To accomplish this, the product need not necessarily be taken out of the mixture bodily; it sufllces to convert it, in situ, to some other molecule which will not react.

The equations for the reactions involved in this application, using structural formulae, are as follows:

equilib- NHfl'CHrCHaCOr-kOH' (beta-alanine) (hydroxide (anion 0! any (water) ion) salt oibeta-alanine) C i H (anion of any salt oibeta-alanine) (alpha-hydroxy-bem.

beta-dimethy butyrolactone CHr-O 0H CHg-C-GH l-NH CHICHIC 0:-

(s postulated intermediate) Equation 1 is an equilibrium known to exist mainly in the form of its products. By it betaalanine, which will not react with the lactone, is converted to the anion of one of its salts. To iumish the hydroxide ion, any water-soluble strong alkaline base may be used, including sodium, potassium and lithium; or an alkaline earth base such as barium hydroxide.

Equation 2 is an equilibrium reaction in which, it lactone and anion oi beta-alanine are mixed in equimolecular proportions, titty percent of the beta-alanine remains unchanged, as determined by the Borensen iormol titration.

Equation 3 is apparently a reaction in which the equilibrium exists at a point where all or nearly all of the mixture exists as product, and little, if any, exists as reactants.

Equation 4 is of the same time as Equation 3.

when the solution or sodium hydroxide, betaalanine, and the lactone is mixed, all four equilibria are set up. Notice that Equation 2 uses up one 01' the products (the anion oi beta-alanine) of Equation 1. This would tend to shift the equilibrium in Equation 1 to the right. However, Equation 4, which goes on simultaneously, uses up one of the reactants (the hydroxide ion, OH) of Equation 1, thus having the reverse effect.

But if the Reaction 1 is reversed. note that that means that the beta-alanine anion is converted back to beta-alanine. and that means it is taken out as reactant in Equation 2, and that that reaction is reversed.

This is counterbalanced by the fact that in 5 Reaction 3 the equilibrium point lies so far to the right that {or all practical purposes it may be said that the reaction is irreversible, and that as soon as any pantothenic acid anion is formed the reactants which go into making it are essentially removed from the solution.

Hence, there are two forces acting on the betaalanine anion: Equation 3 pulls it through the intermediate product to form pantothenic acid anion; and Equation 4 pulls it back through Equation 1 to form beta-alanine. All this takes about an hour. At the end of this hour, it equimolecular amounts of the three reagents have been taken, there are present:

(a) Anion oi pantothenic acid (b) Anion of alpha, gamma-dihydroxy-beta,

beta-dimethyl butyric acid (0) Beta-alanine (d) The water used as solvent.

By experiment it was found that, after an hour, (a) (b) and (0) were present in equal amounts, 1. e., half the material went through the Reactions 1, 2 and 3, and the other half went through Reaction 4.

Now suppose a large excess of lactone, such as three molecules for one of each of the other reagents, is used. The first effect is that the excess of lactone shifts the equilibrium of Equation 2 to the right, so that only forty-five per cent of the beta-alanine anion remains, and at the end of one hour there are now present, for each molecule of beta-alanine originally used:

(a) Anion of pantothenic acid mol 0.55

(e) Solvent water.

. equilibria.

Since (a) and (b) are to all intents and purposes irreversibly formed, the situation isthe same as if they were not present, with respect to the other There is now lactone and beta-alanine present in the ratio of 2 mols to 0.45 mol, or 4:1, but there is no hydroxide ion (011) left. Therefore, 0.45 mol of 011- (the amount equivalent to the unreacted beta-alanine) is now added, and the whole cycle is repeated, with the net result of increasing the amounts of (a) and (2)) present, and decreasing the amounts of (c) and (d).

It might be supposed that since, in the process, the ratio of lactone to unreacted beta-alanine is continually increasing, the percentage of the remainder of the beta-alanine converted each time would also increase. However, this is more than counterbalanced by the fact that the concentration of beta-alanine is steadily diminishing, so that the hydroxide ion has an increasingly greater opportunity for reacting with the lactone instead, since the rate of decrease of the concentration of the latter is much less; hence, diminishing amounts of beta-alanine are converted by the repetition of the process.

Pantothenic acid preparations made by the process outlined in this application have physiological or vitamin-like activity for species other than chicks. For example, Dr. F. Daft and Dr. W. H. Sebrell, of the National Institute of Health at Washington, D. C., have found that such a preparation was effective in preventing or curing degeneration of the adrenal glands in rats. Vitamin-like activity of pantothenic acid may also be expected in the case of other species. Experimental work on this question is in progress in various laboratories in this country. The reference specifically to chicks in this application is due to the fact that this species is convenient for the biological assay of substances which have pantothenic acid activity. Once such activity has been established by means of the biological assay with chicks, the use of the preparation for other species may be expected, under the correct conditions of use, to result in therapeutic or vitamin-like action.

Of the amino acids employed, beta-alanine has been specifically referred to, but other forms may be used if closely related, such as beta-amino butyric acid. Derivatives of beta-alanine which leave it with an NHz instead of an NH: group may be employed. This would include the following:

O NH: CH:CH O CH3 Methyl ester 0 l NHzCHgCHz-O CHQC H3 Ethyl ester Isopropyl ester NHgCHzCHzL-OCHgCaHs Benzyl ester and amides such as beta-alanyl amide and other commonly recognized derivatives.

Alpha-hydroxy-beta, beta-dimeth'ylbutyrolactone has also been specifically referred to, especially the di form thereof, but the dor lform may also be employed, and related lactones such as butyrolactone and alpha-hydroxy-butyrolactone. Similarly, any water-soluble base may be used, such as potassium, sodium, or barium hydroxide, A 1:1:3 ratio of the ingredients employed has been stated, but other ratios are possible, such as 1:1:2, 1:1:4, etc., and the solution of sodium hydroxide may be increased in strength, as a 3.3 normal solution has been specified.

It is desired to point out that the salts of pantothenic acid may be most suitably prepared by intimately mixing at relatively low temperatures, as for example, less than 25 C. and preferably nearer 0 C. or at least between 0 C. and 10 0., aqueous solutions of a lactone and a salt of beta-alanine. The positive metal constituent of the beta-alanine salt being preferably, but not necessarily selected from the alkali and the alkaline earth metals. The lactone is preferably selected from a group consisting of l-, (11- and dalpha hydroxy beta, beta dimethylbutyrolactones. When these constituents react there is at once produced a salt of pantothenic acid corresponding to the salt of the beta-alanine selected in yields which are greatly influenced by the molar ratio of the lactone to the beta-alanine salt as previously pointed out, and also influenced to a substantial extent by the concentration of the solutions which are mixed.

In the preferred form of the invention, the

lactone solution comprises ninety percent lactone and the beta-alanine salt solution is 10 normal. If the molar ratio between the lactone and the beta-alanine is about 2:1, the yield of the salt of pantothenic acid is in the neighborhood of fifty-five percent. It is of course, desired to state that the above is set forth by way of illustration, and not by way of limitation.

It is desired to point out that that portion of the beta-alanine salt which does not form the salt of pantothenic acid reacts with the unchanged lactone present to produce the corresponding salt of alpha-gamma-dihydroxy-beta, beta-dimethyl butyric acid and free beta-alanine. This side reaction proceeds at a much slower rate than the main reaction, but is usually completed at the end of a half hour.

As herein pointed out, it has been discovered that if an amount, equivalent to the amount of free beta-alanine formed, of a material furnishing a hydroxyl ion either dry or in solution, is added to the original reaction mixture, there is a further coupling between the in situ regenerated beta-alanine salt and the unreacted lactone whereby further quantities of the salt of pantothenic acid are formed and a smaller amount of free beta-alanine is left. Preferably, although not necessarily, the material furnishing the hydroxyl ion is an alkali or alkaline earth oxide or hydroxide.

After the reaction proceeds for about a period of two hours, a still additional quantity of a water-soluble base is added to the so-treated reaction mixture, said water soluble base functioning to cause a further coupling between the remaining regenerated beta-alanine salt and the unreacted lactone. Here, again, the further addition may be an alkali or alkaline earth compound, as for example, oxide or hydroxide. The addition of further quantities of basic-forming metal or hydroxide may be continued, but does not materially increase the yield.

In the most satisfactory form of producing the salt of pantothenic acid, it is desirable to use as one of the reacting ingredients, the potassium salt of beta-alanine, since solutions of high concentration are quite readily obtained by mixing beta-alanine with 10 normal potassium hydroxide.

Lesser concentrations down to 1 normal may be used, but tend to produce smaller yields. Greater concentrations at 10 normal may also be used but the reacting solution becomes viscous and is less easily stirred.

It is desired to point out that it is highly desirable when it is desired to increase the yield of the salt of pantothenic acid, to provide an excess of lactone relative to the beta-alanine in order to drive the equilibrium involved during the reaction period towards the formation of an increased amount of the salt of pantothenicacid. Most satisfactory results are produced when the ratio of the lactone to beta-alanine salt is about two mols of lactone to one of the beta-alanine salt. Other ratios may be used. Greater ratios tov some extent increase the yield, but not appreciably, and lesser ratios are not as effective.

It is desired to point out that the desired eilfect of increasing the yield of the salt of pantothenic acid may be accomplished by having present in the reaction mixture an excess of beta-alanine salt instead of an excess of the lactone. However, since it is easier to recover the excess lactone than the excess beta-alanine salt, the preferred procedure for carrying out the reaction is to have the lactone present in excess.

The following is an additional example of a satisfactory method of producing salts of pantothenic acid employing the present invention:

89 g. (1 mol) of beta-alanine is dissolved in 100 cc. of ten normal potassium hydroxide. 260 g. (2 mol) of alpha-hydroxy-beta, beta-dimethylbutyrolactone is dissolved in 26 cc. of water. The two solutions are chilled to a temperature between and C. They are then added together with stirring. At one hour intervals, the two successive portions of ten normal potassium hydroxide are added, the first 50 cc., the second cc. At the end of three hours, the solutions is treated by the slow addition of 100 cc. of chilled 18 N sulfuric acid in such a manner that the temperature does not rise above 25 C. The solution is then added to five volumes of acetone, the precipitated sulfates removed by filtration, the acetone removed by distillation, and the resulting solution neutralized with calcium carbonate. The resulting solution is filtered to remove the excess calcium carbonate, concentrated in vacuo to a heavy syrup, and taken up in two volumes of methanol. This solution is then introduced into ten times its volume of acetone. The precipitated calcium pantothenate is filtered, washed with fresh acetone and dried in a vacuum drier. The yield is about 215 g., or ninety per cent of the theoretical amount. From the filtrate the solvents are removed and the excess lactone recovered in good yield.

In the above example, the pantothenic acid is isolated in the form of the calcium salt, although an alkali salt is formed during the reaction treatment. It is desired to point out that it is highly desirable to obtain the final product in the form of a calcium salt because of the ease of handling calcium pantothenate in the dry form.

The present application is a division of copending application Serial No. 346,454, filed July 19, 1940. i

What is claimed is:

l. The method of producing pantothenic acid comprising reacting at a temperature below 25 C. an aqueous solution of beta-alanine, a base in the proportion of about 1 mole for each mole asvaass or beta-alanine, the metal component of said base being selected from the group consisting of alkali and alkaline earth metals. and alpha-hydroxy-beta, beta-dimethylbutyrolactone, the molar ratio of the lactone to the beta-alanine varying from about 2: 1 to about 4:1, periodically adding to the reaction product a solution or a water-soluble alkali base. and recovering the pantothenic acid-content from the resulting reaction mass.

2. The method of producing pantothenic acid comprising reacting at a temperature below about 25 C., an aqueous solution of a metal salt of betaalanine, the metal component of which is selected from the group consisting of alkali and alkaline earth metals, with alpha-hydroxy-beta, beta-dimethylibutyrolactone, the molar ratio of the lactone to the beta-alanine salt being at least 2:1, periodically adding to the reaction product a solution of a water-soluble alkali 'base, and recovering the pantothenic acid-content from the resulting reaction-mass.

3. The method of producing pantothenic acid comprising reacting at a temperature below 25 C. an aqueous solution of beta-alanine, a base in the proportion of about 1 mole for each mole of beta-alanine, the metal component of said base being selected from the group consisting of alkali and alkaline earth metals, and alpha-hydroxy-beta, beta-dimethylbutyrolactone, the molar ratio of the lactone to the beta-alanine varying from about 2:1 to about 4: 1, periodically adding to the reaction product a solution of a water-soluble alkali hydroxide, and recovering the antothenic acid-content from the resulting reaction-mass.

4. The method of producing pantothenic acid comprising reacting at a temperature below about 25 C., an aqueous solution of a metal salt of beta-alanine, the metal component of which is selected from the group consisting of alkali and alkaline earth metals, with alpha-hydroxy-beta, beta-dimethylbutyrolactone, the molar ratio of the lactone to the beta-alanine salt being at least 2:1,-periodically adding to the reaction product a solution of a water-soluble alkali hydroxide, and recovering the pantothenic acid-content from the resulting reaction-mass.

5. The method of producing pantothenic acid comprising reacting at a temperature below 25 C. an aqueous solution of beta-alanine, a base in the proportion of about 1 mole for each mole of beta-alanine. the metal component of said base being selected from the group consisting of alkali and alkaline earth metals, and alphahydroxy-beta, beta-dimethylbutyrolactone, the molar ratio of the lactone to the beta-alanine varying from about 2:1 to about 4:1, periodically adding to the reaction product a solution of a water-soluble alkali base having a strength varying between about 1 and about 10 normal, and recovering the pantothenic acid-content from the resulting reaction mass.

6. The method of producing pantothenic acid comprising reacting at a temperature below about 25 C., an aqueous solution of a metal salt of beta-alanine, the metal component of which is selected from the group consisting of alkali and alkaline earth metals, with alpha-hydroxy-b'eta, beta-dimethylbutyrolactone, the molar ratio of the lactone to the beta-alanine salt being at least 2:1, periodically adding to the reaction product a solution of a water-soluble alkali base having a strength varying between about 1 and about 10 normal, and recovering the pantothenic acidcontent from the resulting reaction-mass.

7. The method of producing pantothenic acid comprising reacting at a temperature varying from about 0 C. to about 10 C., an aqueous solution of betaalanine, a base in the proportion of about 1 mole for each mole of [beta-alanine, the metal component of said base being selected from the group consisting of alkali and alkaline earth metals, and alpha-hydroxy-beta. beta-dimethylb'utyrolactone, the molar ratio of the lactone to the beta-alanine varying from about 2:1 to about 4:1, periodically adding to thereaction product a solution or a water-soluble alkali base, and recovering the pantothenic acid-content from the resulting reaction mass.

SIDNEY H. BABOOCK. J1. 

